The present invention is related generally to assays and more specifically to assays which determine compounds which might provide a therapeutic effect of a disease associated with a conformationally altered protein.
There are a considerable number of diseases associated with a conformationally altered protein. For example, Alzheimer""s disease is associated with APP, Axcex2 peptide, xcex11-antichymotrypin, tau and non-Axcex2 component. Many of these diseases are neurological diseases. However, type II Diabetes is associated with Amylin and Multiple myeloma-plasma cell dyscrasias is associated with IgGL-chain. The relationship between the disease onset and the transition from the normal protein to the conformationally altered protein has been examined very closely in some instances such as with the association between prion diseases and PrPSc.
Prion diseases are a group of fatal neurodegenerative disorders that can occur in hereditary, sporadic, and infectious forms (Prusiner, S. B. Scrapie prions. Annu. Rev. Microbiol. 43, 345-374 (1989)). These illnesses occur in humans and a variety of other animals (Prusiner, S. B. Prions. Proc. Natl. Acad. Sci. USA 95, 13363-13383 (1998)). Prions are infectious proteins. The normal, cellular form of the prion protein (PrP) designated PrPC contains three xcex1- helices and has little xcex2- sheet; in contrast, the protein of the prions denoted PrPSc is rich in xcex2-sheet structure. The accumulation of PrPSc in the central nervous system (CNS) precedes neurologic dysfunction accompanied by neuronal vacuolation and astrocytic gliosis.
The spectrum of human prion diseases includes kuru (Gajdusek, D. C., Gibbs, C. J., Jr. and Alpers, M. Experimental transmission of a kuru-like syndrome to chimpanzees. Nature 209, 794-796 (1966)), Creutzfeldt-Jakob disease (CJD) (Gibbs, C. J., Jr., et al. Creutzfeldt-Jakob disease (spongiform encephalopathy): transmission to the chimpanzee. Science 161, 388-389 (1968)), Gerstmann-Strxc3xa4ussler-Scheinker disease (GSS) and fatal familial insomnia (FFI) (Goldfarb, L. G., et al. Fatal familial insomnia and familial Creutzfeldt-Jakob disease: disease phenotype determined by a DNA polymorphism. Science 258, 806-808 (1992); Medori, R., et al. Fatal familial insomnia: a second kindred with mutation of prion protein gene at codon 178. Neurology 42, 669-670 (1992)), and a new form of human prion disease, new variant CJD (nvCJD), which has emerged in Great Britain and France (Will, R. G., et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347, 921-925 (1996); Cousens, S. N., Vynnycky, E., Zeidler, M., Will, R. G. and Smith, P. G. Predicting the CJD epidemic in humans. Nature 385, 197-198 (1997); Will, R. G., et al. Deaths from variant Creutzfeldt-Jakob disease. Lancet 353, 979 (1999)). Several lines of evidence have suggested a link between the nvCJD outbreak and a preceding epidemic of bovine spongiform encephalopathy (BSE) (Will, R. G., et al. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347, 921-925 (1996); Bruce, M. E., et al. Transmissions to mice indicate that xe2x80x98new variantxe2x80x99 CJD is caused by the BSE agent. Nature 389, 498-501 (1997); Hill, A. F., et al. The same prion strain causes vCJD and BSE. Nature 389, 448-450 (1997); Lasmxc3xa9zas, C. I., et al. BSE transmission to macaques. Nature 381, 743-744 (1996)). Although it is too early to predict the number of nvCJD cases that might eventually arise in Great Britain and elsewhere (Cousens, S. N., Vynnycky, E., Zeidler, M., Will, R. G. and Smith, P. G. Predicting the CJD epidemic in humans. Nature 385, 197-198 (1997)), it is clear that effective therapeutics for prion diseases are urgently needed. Unfortunately, although a number of compounds including amphotericins, sulfated polyanions, Congo red dye, and anthracycline antibiotics have been reported as prospective therapeutic agents (Ingrosso, L., Ladogana, A. and Pocchiari, M. Congo red prolongs the incubation period in scrapie-infected hamsters. J. Virol. 69, 506-508 (1995); Tagliavini, F., et al. Effectiveness of anthracycline against experimental prion disease in Syrian hamsters. Science 276, 1119-1122 (1997); Masullo, C., Macchi, G., Xi, Y. G. and Pocchiari, M. Failure to ameliorate Creutzfeldt-Jakob disease with amphotericin B therapy. J. Infect. Dis. 165, 784-785 (1992); Ladogana, A., et al. Sulphate polyanions prolong the incubation period of scrapie-infected hamsters. J. Gen. Virol. 73, 661-665 (1992)), all have demonstrated only modest potential to impede prion propagation, and none have been shown to effect the removal of pre-existing prions from an infected host.
The PrP gene of mammals expresses a protein which can be the soluble, non-disease form PrPC or be converted to the insoluble, disease form PrPSc. PrPC is encoded by a single-copy host gene [Basler, Oesch et al. (1986) Cell 46:417-428] and when PrPC is expressed it is generally found on the outer surface of neurons. Many lines of evidence indicate that prion diseases result from the transformation of the normal form of prion protein (PrPC) into the abnormal form (PrPSc). There is no detectable difference in the amino acid sequence of the two forms. However, PrPSc when compared with PrPC has a conformation with higher xcex2-sheet and lower xcex1-helix content (Pan, Baldwin et al. (1993) Proc Natl Acad Sci USA 90:10962-10966; Safar, Roller et al. (1993) J Biol Chem 268:20276-20284). The presence of the abnormal PrPSc form in the brains of infected humans or animals is the only disease-specific diagnostic marker of prion diseases.
PrPSc plays a key role in both transmission and pathogenesis of prion diseases (spongiform encephalopathies) and it is a critical factor in neuronal degeneration (Prusiner (1997) The Molecular and Genetic Basis of Neurological Disease, 2nd Edition: 103-143). The most common prion diseases in animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE) of cattle (Wilesmith and Wells (1991) Curr Top Microbiol Immunol 172:21-38). Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3) Gerstmann-Strxc3xa4ussler-Sheinker Disease (GSS), and (4) fatal familial insomnia (FFI) [Gajdusek (1977) Science 197:943-960; Medori, Tritschler et al. (1992) N Engl J Med 326:444-449]. Initially, the presentation of the inherited human prion diseases posed a conundrum which has since been explained by the cellular genetic origin of PrP.
The assembly and misassembly of normally soluble proteins into conformationally altered proteins is thought to be a causative process in a variety of other diseases. Structural conformational changes are required for the conversion of a normally soluble and functional protein into a defined, insoluble state. Examples of such insoluble protein include: Axcex2 peptide in amyloid plaques of Alzheimer""s disease and cerebral amyloid angiopathy (CAA); xcex1-synuclein deposits in Lewy bodies of Parkinson""s disease, tau in neurofibrillary tangles in frontal temporal dementia and Pick""s disease; superoxide dismutase in amyotrophic lateral sclerosis; huntingtin in Huntington""s disease; and prions in Creutzfeldt-Jakob disease (CJD): (for reviews, see Glenner et al. (1989) J. Neurol. Sci. 94:1-28; Haan et al. (1990) Clin. Neurol. Neurosurg. 92(4):305-310).
Often these highly insoluble proteins form aggregates composed of nonbranching fibrils with the common characteristic of a xcex2-pleated sheet conformation. In the CNS, amyloid can be present in cerebral and meningeal blood vessels (cerebrovascular deposits) and in brain parenchyma (plaques). Neuropathological studies in human and animal models indicate that cells proximal to amyloid deposits are disturbed in their normal functions (Mandybur (1989) Acta Neuropathol. 78:329-331; Kawai et al. (1993) Brain Res. 623:142-6; Martin et al. (1994) Am. J. Pathol. 145:1348-1381; Kalaria et al. (1995) Neuroreport 6:477-80; Masliah et al. (1996) J. Neurosci. 16:5795-5811). Other studies additionally indicate that amyloid fibrils may actually initiate neurodegeneration (Lendon et al. (1997) J. Am. Med. Assoc. 277:825-31; Yankner (1996) Nat. Med. 2:850-2; Selkoe (1996) J. Biol. Chem. 271:18295-8; Hardy (1997) Trends Neurosci. 20:154-9).
In both AD and CAA, the main amyloid component is the amyloid xcex2 protein (Axcex2). The Axcex2 peptide, which is generated from the amyloid xcex2 precursor protein (APP) by two putative secretases, is present at low levels in the normal CNS and blood. Two major variants, Axcex21-40 and Axcex21-42, are produced by alternative carboxy-terminal truncation of APP (Selkoe et al.(1988) Proc. Natl. Acad. Sci. USA 85:7341-7345; Selkoe, (1993) Trends Neurosci 16:403-409). Axcex21-42 is the more fibrillogenic and more abundant of the two peptides in amyloid deposits of both AD and CAA. In addition to the amyloid deposits in AD cases described above, most AD cases are also associated with amyloid deposition in the vascular walls (Hardy (1997), supra; Haan et al. (1990), supra; Terry et al., supra; Vinters (1987), supra; Itoh et al. (1993), supra; Yamada et al. (1993), supra; Greenberg et al. (1993), supra; Levy et al. (1990), supra). These vascular lesions are the hallmark of CAA, which can exist in the absence of AD.
Human transthyretin (TTR) is a normal plasma protein composed of four identical, predominantly xcex2-sheet structured units, and serves as a transporter of hormone thyroxin. Abnormal self assembly of TTR into amyloid fibrils causes two forms of human diseases, namely senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP) (Kelly (1996) Curr Opin Strut Biol 6(1):11-7). The cause of amyloid formation in FAP are point mutations in the TTR gene; the cause of SSA is unknown. The clinical diagnosis is established histologically by detecting deposits of amyloid in situ in bioptic material.
To date, little is known about the mechanism of TTR conversion into amyloid in vivo. However, several laboratories have demonstrated that amyloid conversion may be simulated in vitro by partial denaturation of normal human TTR [McCutchen, Colon et al. (1993) Biochemistry 32(45):12119-27; McCutchen and Kelly (1993) Biochem Biophys Res Commun 197(2) 415-21]. The mechanism of conformational transition involves monomeric conformational intermediate which polymerizes into linear xcex2-sheet structured amyloid fibrils [Lai, Colon et al. (1996) Biochemistry 35(20):6470-82]. The process can be mitigated by binding with stabilizing molecules such as thyroxin or triiodophenol (Miroy, Lai et al. (1996) Proc Natl Acad Sci USA 93(26):15051-6).
The precise mechanisms by which neuritic plaques are formed and the relationship of plaque formation to the disease-associated neurodegenerative processes are not well-defined. The amyloid fibrils in the brains of Alzheimer""s and prion disease patients are known to result in the inflammatory activation of certain cells. For example, primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar xcex2-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The signaling response elicited by xcex2-amyloid and prion fibrils leads to the production of neurotoxic products, which are in part responsible for the neurodegenerative. C. K. Combs et al, J Neurosci 19:928-39 (1999).
Despite considerable efforts effective therapeutic compounds for the treatment of diseases associated with conformationally altered protein have not been discovered. The present invention offers an assay for identifying therapeutic compounds and further disclose a class of compounds which have been shown to be effective in clearing deposits of conformationally altered proteins associated with disease.